1. health and fitness
2. disease

aj
ROBOSCREEN
®
BetaPrion BSE EIA Test Kit
Test for in vitro purification and detection of BSE PrPres
Within the European Union, this test is approved as rapid test for the BSE testing program
on cattle which is set up in accordance with Regulation (EC) No 999/2001.
Version 3.3/2014
BetaPrion® BSE EIA Test Kit
Cat.No. 0104000102
The BetaPrion® BSE EIA Test Kit consists of two modules, BetaPrion® BSE Purification Kit and
BetaPrion® BSE Detection Kit.
BetaPrion® BSE Purification Kit allows the use of 2 ml tubes or 96 deep well plates.
One BetaPrion® BSE EIA Test Kit contains sufficient reagents to analyse 90 bovine brain samples.
The producer of the rapid tests must have a quality assurance system in place
agreed by the Community reference laboratory, which ensures that the test
performance does not change. The producer must provide the test protocol to the
Community reference laboratory. Sampling tools and modifications to the rapid test
or to the test protocol (including sampling) may only be made following advance
notification to the Community Reference Laboratory (CRL) and provided that the
Community reference laboratory finds that the modification does not reduce the
sensitivity, specificity or reliability of the rapid test. That finding shall be
communicated to the Commission and to the national reference laboratories (Based
on Regulation (EC) No 1053/2003 amending Regulation (EC) No 999/2001).
Abbreviations:
BSE (Bovine spongiform encephalopathy), HRP (Horseradish peroxidase), PrP (Prion protein)
BSA (Bovine serum albumin), TMB (Tetramethylbenzidine); ELISA (Enzyme-linked immunosorbent
assay), RT (Room Temperature), PK (proteinase K)
1
CONTENTS
Page
1.
Introduction
3
1.1.
Brief general remarks
3
1.2.
Short description of the test principle
3
1.2.1. Purification of PrPres
res
3
1.2.2. Detection of PrP
3
2.
Components not provided with the kit
4
3.
BetaPrion® BSE Purification Kit
4
3.1.
Sampling
4
3.2.
Kit components
5
3.3.
Preparation of the reagents
6
3.4.
Test Procedure
6
3.5.
Storage conditions and shelf life of components
7
4.
BetaPrion® BSE Detection Kit
7
4.1.
Kit components
7
4.2.
Preparation of the reagents
7
4.3.
Immunoassay
8
4.4.
Storage conditions and shelf life of components
9
5.
Interpretation of the results
9
6.
Precautions
9
7.
Appendices
10
8.
Warranty notice/Additional general remarks
10
9.
General comments
11
10.
Instruction for the sample preparation using
12
AJ Roboscreen BSE sample syringe
2
1.
Introduction
1.1.
Brief general remarks
Prion diseases or transmissible spongiform encephalopathies are neuro-degenerative diseases
that affect both humans and animals (Prusiner 1998). All prion diseases share the same molecular
pathogenic mechanism that involves conversion of normal cellular prion protein (PrPsen) into a form
that is insoluble in non-ionic detergents and partially resistant to proteases (PrPres) (Pan et al.
1993). Both PrPres and PrPsen are encoded within a single exon of a chromosomal gene coding for
a protein of ~ 250 amino acids (Basler et al. 1986). Many mammalian PrPs have a 22 amino acid
N-terminal signal sequence (Hope et al. 1986; Turk et al. 1988) and 23 amino acid C-terminal
signal sequence encoding for attachment of a glycosylphosphatidyl-inositol anchor (Stahl et al.
1987, 1990). The mature protein of 209 amino acids contains one disulfide bond (Turk et al. 1988)
and has two sites of asparagine-linked glycosylation (Endo et al. 1989; Oesch et al. 1995).
1.2.
Short description of the test principle
The BetaPrion® BSE EIA Test Kit is a continuous 100 -110 min test for the detection of PrPres in
bovine brain samples. Specimens of bovine brain are homogenized and incubated with
Proteinase K. Solubilzed PrPres is captured by a specific monoclonal anti-PrP antibody coated to
the wells of a microtitre strip. Bound PrPres is detected with a HRP-conjugated anti-PrP
antibody. The wells are washed and a substrate solution is added. The developed colour
indicates the existence of PrPres in the specimen in comparison to a negative and a positive
control in case the sample OD is greater than the declared cut-off.
1.2.1. Purifcation of PrPres
adding of PK solution
and digestion.
adding of precipitation
solution and
concentration of PrPres
by centrifugation
heating of PrPres for
solubilisation
1.2.2. Detection of PrPres
+
HRP conjugated
anti-PrP antibody
PrPres
H2O2 + TMB
O.D. 450/620 nm
HRP conjugated
anti-PrP antibody
45 min
PrPres
Microtiter plate coated with
anti-PrP capture antibody
anti-PrP capture antibody
3
2. Components not provided with the kit
For the Purification kit
• Homogenisation device, Ribolyzer, FastPrep®, FastPrep® 24 System (MP Biomedicals
USA) or Precellys®24 (Bertin France)
• BSE sample syringes
• 15 or 50 ml tubes, e.g. Falcon tubes, for the dilution of Proteinase K
• 2 ml tubes, e.g. Eppendorf (Germany)
• 96 ml deep well plates, e.g. Brand (Germany)
• Single and multi channel pipetting tools (adjustable, 100-1000 µl, 10-100 µl) and tips
• Desktop centrifuge (16.000g or 3.200 g), e.g. 5415D or 5815 (Eppendorf Germany)
• Thermostate, e.g. Thermomixer comfort (Eppendorf Germany) and heating block for 96
deep well plates, e.g. AJ Cybertron GmbH (Germany)
• 96 ml deep well plate aspiration tool, e.g. Washer (Kisker Germany)
• Syringe, e.g. 1ml and blunt end needles, 25Gx1.5’’ (e.g. Agesa Germany)
• Vortex mixer, e.g. Reax (VWR International Germany)
• Refrigerator 4-8°C
For the Detection Kit
• Single channel pipetting tools (adjustable, 100-1000 µl, 10-100 µl) and tips
• 15 ml tubes, e.g. Falcon tubes for the dilution of reagents
• Multichannel pipette (adjustable, 30-300 µl)
• Microplate reader, Anthos Reader 2001 (Anthos Labtec Instruments GmbH Austria)
or CM Sunrise reader (Tecan Deutschland GmbH Germany)
• Microplate washer, e.g. CM Columbus washer (Tecan Deutschland GmbH
Germany)
3. BetaPrion® BSE Purification Kit
3.1.
Sampling
Bovine brain of the obex region (Figure 1) must be used as starting material for the
BetaPrion® BSE EIA Test Kit.
Note: After sample collection, a complete hemi-section of the brain stem with an intact obex
region must remain available for confirmatory testing.
Sampling and laboratory testing must follow the Regulation (EC) No 999/2001 Chapter C
which refers in terms of collection of samples to the latest edition of the “Manual of Standards for
Diagnostic Tests and Vaccines of the International Office of Epizootic Diseases (OIE)) stating:
“The preferred sample for immunoassay should be at, or as close to the obex as possible, but
no further than 1.5 cm anterior to the obex.” Figure 1 shows the sampling area, either the left or
the right hand side can be used.
4
sampling sites
Obex Spinal Cord
Figure 1: Sampling area
Two opportunities for sampling:
(1) Cut 350 ± 50 mg nervous tissue from this area from the left or the right side of the
brainstem with a scalpel and weigh the sample to ensure the correct amount.
(2) Sample preparation using the AJ Roboscreen BSE sample syringe according to the
instruction under (10) on page 14.
3.2.
Kit components
Short name
Homogenisation tubes
P1
5
Type/content
Quantity
Transparent tubes (2 ml) with red cap
96 tubes
containing Homogenisation buffer (1.3
ml) and keramic beads, ready to use.
Store at RT.
Proteinase K buffer in transparent bottle 1 x bottle
with red label. Store at RT.
(24 ml)
P2
Proteinase K solution in transparent
tube with yellow cap. Store at 4-8°C.
1 x tube
(0.6 ml)
P3
Precipitation solution in brown bottle
with red label containing detergents,
ready to use. Store at RT.
1 x bottle
(45 ml)
P4
Solubilisation buffer in transparent
bottle with red label containing
detergent solution, ready to use. Store
at RT.
1 x bottle
(4 ml)
3.3.
Preparation of the Reagents
Ready to use reagents:
The homogenisation tubes, precipitation solution (P3) and the solubilisation buffer (P4) are
ready to use reagents.
Reconstituted reagents:
Proteinase K buffer (P1) is required for the dilution of Proteinase K (P2) and must be used as
follows:
Number of
samples
8
40
60
90
2 ml tube purification
P1 buffer
(ml)
2.0 ± 0.20
10.0 ± 1.00
15.0 ± 1.50
20.0 ± 2.00
PK solution
(µl)
50.0 ± 5.00
250.0 ± 25.00
375.0 ± 37.50
500.0 ± 50.00
96 deep well plate purification
P1 buffer
(ml)
1.0 ± 0.10
5.0 ± 0.50
7.5 ± 0.75
10.0 ± 1.00
PK solution
(µl)
50.0 ± 5.00
250.0 ± 25.00
375.0 ± 37.50
500.0 ± 50.00
3.4. Test Procedure
Place the brain sample (350 ± 50 mg) into a homogenisation tube and homogenize for 45 sec at
maximum speed (6.5) in FastPrep® or at 5500-5600 rpm in Precellys®24, respectively.
Homogenized brain samples can be stored at –20 ± 2 °C for up to 12 months. For further use, thaw
the samples at RT and vortex for 2 sec at maximal speed. Samples can be submitted to a
maximum of 3 freezing-thawing cycles.
2 ml tube purification
96 deep well plate purification
Transfer 200 ± 20 µl of homogenized
sample with a 1-ml syringe using a fine
needle (blunt end needle 25Gx1.5’’) to a
new 2 ml Eppendorf tube.
Add 200 ± 20 µl of the reconstituted
Proteinase K solution, mix by vortexing (2
sec at maximum speed) or inverting the
tubes 3-6 times.
Incubate at 37± 1 °C for 15 ± 1.5 min with
continuous shaking (750 rpm).
Add 400 ± 40 µl precipitation solution, mix
by vortexing (2 sec at maximal speed) or
inverting the tubes 3-6 times and incubate
the sample for 15 ± 1.5 min at RT.
Centrifugate16.000 ± 1.600 g for 5 ± 0.5
min. Discard the supernatant carefully and
removed the remaining supernatant by
tipping the tubes on a paper towel.
Transfer 300 ± 30 µl of homogenized
sample with a 1-ml syringe using a fine
needle (blunt end needle 25Gx1.5’’) to 90
well of the 96 deep well plate.
Add 100 ± 10 µl of the reconstituted
Proteinase K solution, mix by pipetting or
inverting the sealed plate 3 times.
Add 25 ± 2.5 µl solubilisation buffer to the
pellet and heat at 99 ± 1°C for 7 ± 0.5 min
with intervall shaking (1200-1500 rpm).
Cool down the tubes for at least 5 min at
RT. Samples can be stored up to 6 h at 48°C or at RT.
Incubate at 37± 1 °C for 15 ± 1.5 min with
continuous shaking (300 rpm).
Add 400 ± 40 µl precipitation solution, mix
by pipetting or inverting the selaed plate 3
times and incubate the sample for 15 ± 1.5
min at RT.
Centrifugate 3.200 ± 320 g for 15 ± 1.5 min.
Discard the supernatant carefully and
removed the remaining supernatant by
tipping the plate on a paper towel or
aspirate the supernatant by pipetting tools.
Add 25 ± 2.5 µl solubilisation buffer to the
pellet and heat at 99 ± 1°C for 7 ± 0.5 min
with intervall shaking (1200-1500 rpm).
Cool down the plate for at least 5 min at RT.
Samples can be stored up to 6 h at RT.
6
Analyze the sample for PrPres protein using the BetaPrion® BSE Detection Kit.
3.5.
Storage conditions and shelf life of the components
Store all components exceptional Proteinase K at room temperature (15-25°C). The tube
containing Proteinase K has to be stored at 4-8°C. The guaranteed shelf life of the reagents is
12 months. The reconstituted Proteinase K solution is stable up to 3 hours at room temperature
(15-25 °C) or up to 8 hours at 4-8 °C.
4. BetaPrion® BSE Detection Kit
4.1.
Kit components
Short name
Type/content
Quantity
D1
Immunostrips coated with monoclonal anti-PrP capture
antibody, stabilized, ready-to-use.
1 immunoplate
(12 x 8 wells)
D2
Washing buffer (10x concentrate) in a transparent bottle,
containing 0.01 % Na-Merthiolat as preservative.
1 x bottle
(100 ml)
D3
Positive control in transparent tubes (2 ml) with blue caps 2 x tubes
containing lyophilized recombinant bovine prion protein.
D4
Negative control in transparent tubes with white caps,
ready-to-use.
3 x tubes
(3 x 0.1 ml)
D5
Horseradish peroxidase conjugate in amber bottle with
black cap containing 8x concentrate of conjugated antiPrP antibody. Preservative: 0.1 % Proclin 300.
Dilution buffer in transparent bottle containing buffered
saline solution with BSA, ready to use. Preservative: 0.1
% Proclin 300.
Tetramethylbenzidine solution in amber bottle with red
cap containing a liquid stabilized TMB solution.
1 x bottle
(2.5 ml)
D8
Peroxide substrate solution in amber bottle with green
cap containing peroxide solution.
1 x bottle
(1 ml)
D9
Staining buffer in transparent bottle containing buffered
saline solution.
1 x bottle
(20 ml)
D10
Stop solution in transparent containing 1 M sulphuric
acid, ready-to-use.
Sealing tape
1 x bottle
(25 ml)
1 x tape
D6
D7
4.2.
1 x bottle
(20 ml)
1 x bottle
(1 ml)
Preparation of the reagents
Allow immunostrips, washing buffer, dilution buffer, staining buffer and stop solution to
equilibrate to room temperature prior performing the assay.
Ready to use reagents:
After prewarming to room temperature of the immunostrips (D1), open the bag with a scissors
and remove the needed strips. Dilution buffer (D6) and Stop solution (D10) are ready to use
reagents.
7
Reconstituted reagents:
Washing solution (D2):
Dilute the 10 x concentrated wash buffer with distilled water (add 900 ml of aqua dest to the 100
ml concentrate) before the washing step of the immunoassay.
Positive control (D3):
Add 0.5 ml of diluted HRP conjugate to positive control tube and vortex for 2 sec at the
beginning of the immunoassay.
Negative control (D4) :
Add 0.5 ml of diluted HRP conjugate to negative control tube and vortex for 2 sec at the
beginning of the immunoassay.
HRP conjugate (D5) :
Dilute the 8x HRP conjugate with dilution buffer (for 1 plate add 14 ml dilution buffer to 2 ml of
conjugate) for diluted HRP conjugate.
Staining solution (D7 + D8 + D9)
Mix 12 ml of staining buffer (D9) with 300 µl of TMB solution (D7) and 180 µl peroxide substrate
solution (D8) during the washing step of the immunoassay.
4.3.
Immunoassay
Assay procedure for using 1 immunoplate (12x8 strips). If you use only individual strips please
recalculate the reagent volumes required accordingly. For pipetting into the wells of the
immunoplate please follow the pipetting scheme (chapter 7)
1. Add 125 ± 12.5 µl of diluted HRP conjugate to the solubilized sample resulted from the
BetaPrion® BSE Purification Kit step 9 and mix it by 3 times pipetting or inverting or
vortexing for 2 sec.
2. Pipet 4x 100 ± 10 µl of one negative and 2x 100 ± 10 µl of one positive control,
respectively.
3. Pipet 100 ± 10 µl of the diluted sample per well of the coated immunostrips.
4. Cover the strips with sealing tape and incubate for 45 ± 4.5 min at RT.
5. Remove the sealing tape and wash the strips 5 times with 200-300 ± 20-30 µl diluted
wash buffer either manually or by using the Columbus washer program BSE-5*.
6. Prepare the staining solution.
7. Dispense 100 ± 10 µl of prepared staining solution per well.
8. Incubate for 10 ± 1 min in the dark at room temperature (for this step do not seal the
plate with the sealing tape).
9. Terminate reaction by adding 150 ± 15 µl of stop solution per well.
10. Read the O.D. at 450 nm and 620 nm as reference wave length using the microplate
reader within 15 minutes after termination of the reaction.
*) Columbus washer program BSE-5 is a plate washing program using the overflow modus with 5 x 1000 µl
washing buffer and an aspiration cycle at the end of the program.
8
4.4.
Storage conditions and shelf life of reagents
Reconstituted reagents have a shelf life as follows:
Short name
Reagent
Shelf life
D1
Coated immunostrips after
opening of the bag.
1 week at 4-8°C
D2
1X washing solution.
1 week at RT
D5
Diluted HRP conjugate
4 h at 4-8°C
D8
Mixed staining solution.
2 h at RT in dark vials
Store all reagents of the Detection kit at 4-8°C. The guaranteed shelf life of the reagents is 12
months.
5.
Interpretation of the results
The cut-off value is defined as 0.2 optical density OD 450/620 nm and must be used for the
discrimination of BSE positive samples from negative samples. The interpretation of data is
done as follows:
-
All samples with an OD 450/620 nm below 0.2 are classified BSE negative.
Samples with an OD 450/620 nm ≧ 0.2 are classified initially reactive and must be re-tested
in duplicate using the original homogenate. If one of the two duplicate readings has an
OD 450/620 nm ≧ 0.2 the sample is classified BSE positive and must be handled according
to the respective national guidelines.
Samples and the corresponding tissue giving positive or inconclusive rapid test results should
be sent to the NRL for confirmation.
Validation of the test
The OD 450/620 nm value of the positive control must be at least 1.0 and of the OD 450/620 nm value of
the negative control must be below. 0.1. The plate must be repeated, when two of the negative
controls have an OD 450/620 nm ≧ 0.1.
6.
Precautions
Prions are a unique class of pathogens which exhibits unusual resistance to conventional
chemical and physical decontamination methods. A Biosafety level 3 laboratory is necessary for
testing of BSE material. National legal regulations have to be considered. The quality of results
generated with BetaPrion® BSE EIA Test Kit depends on compliance with the good Laboratory
Practices (GLP).
Use disposible consumable supplies whenever possible. For decontamination of BSE
positive material soak instruments in 2.5% sodium hypochlorite or 1 M NaOH for at least 1 hour
or clean contaminated surfaces with those solutions. Liquids and waste should be
decontaminated by autoclaving at 134°C for 1 hour.
9
7. Appendices
Pipetting scheme for the coated Microstrips
N
3
11 19 27 35 43 51
N
4
12 20 28 36 44 52
N
5
13 21 29 37 45 53
N
6
14 22 30 38 46 54
P
7
15 23 31 39 47 55
P
8
16 24 32 40 48 56
1
9
17 25 33 41 49 57
2
10 18 26 34 42 50 58
Distribution of the controls and samples:
N
= Negative control
P
= Positive control
1-90 = Samples
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
How to contact aj ROBOSCREEN
If you are using the Internet access our web-site at www.analytik-jena.de.
Please contact us by:
• E-mail at: [email protected],
• Phone at: +49-341-9897340
• or Fax at: +49-341-989734199.
8. Warranty notice/Additional general remarks
During the warranty period the BetaPrion® BSE EIA Test Kit allows precise and reproducible data
recovery combined with excellent sensitivity. For data obtained by violation to the general GLP
guidelines and the manufacturer’s recommendations the right to claim under guarantee is expired.
Safety Statements
-
Homogenisation buffer must not be classified according to the Directive 1999/45/EC.
-
Buffer P1 contains Sodium Dodecylsulfate, which is harmful (Xn) according to the Directive 1999/45/EC. Risk (R)
and Safety Statements (S) are listed below:
S26
= in case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
R22
= harmful if swallowed.
R36/37/38
= irritating to eyes, respiratory system and skin.
S36
= wear suitable protective clothing.
Symbols according to EC No. 1272/2008 (CLP):
-
Buffer P2 contains proteinase K, which is harmful (Xn) according to the Directive 1999/45/EC. Risk (R) and Safety
Statements (S) are listed below:
R42
= may cause sensitization by inhalation.
R43
= may cause sensitization by skin contact.
Symbols according to EC No. 1272/2008 (CLP):
-
Buffer P3 must not be classified according to the Directive 1999/45/EC.
-
Buffer P4 must not be classified according to the Directive1999/45/EC.
10
-
Buffer D1 must not be classified according to the Directive1999/45/EC.
-
Buffer D2 contains Tris[hydroxymethyl]aminomethan, which is irritant (Xi) according to the Directive1999/45/EC.
Risk (R) and Safety Statements (S) are listed below:
R36/38
= irritating to eyes and skin.
Symbols according to EC No. 1272/2008 (CLP):
-
Buffer D3 must not be classified according to the Directive 1999/45/EC.
-
Buffer D4 must not be classified according to the Directive1999/45/EC.
-
Buffer D5 must not be classified according to the Directive1999/45/EG.
-
Buffer D6 must not be classified according to the Directive 1999/45/EC.
-
Buffer D7 contains Tetramethylbenzidine and organic solution, which are toxic and harmful (T, Xn) according to the
Directive 1999/45/EC. Risk (R) and Safety Statements (S) are listed below:
S1
= keep locked up.
S45
= in case of accident or if you feel unwell, seek medical advice immediately.
R25
= toxic if swallowed.
R20/21
= harmful by inhalation and contact with skin
R36
= irritating to eyes
S26
= in case of contact with eyes, rinse immediately with plenty of water and seek. medical advice.
S28
= after contact with skin, wash immediately with plenty of water.
S36
= wear suitable protective clothing.
Symbols according to EC No. 1272/2008 (CLP):
-
Buffer D8 contains peroxide, which is corrosive (C) according to the Directive 1999/45/EC. Risk (R) and Safety
Statements (S) are listed below:
R34
= causes burn
S3
= keep in a cool place
S26
= in case of contact with eyes, rinse immediately with plenty of water and seek. medical advice.
S36/37/39
= wear suitable protective clothing, gloves and eye/face protection.
S45
= in case of accident or if you feel unwell, seek medical advice immediately.
-
Buffer D9 must not be classified according to the Directive 1999/45/EC.
-
Buffer D10 contains Sulphuric acid, which is corrosive (C) according to the Directive 1999/45/EC. Risk (R) and
Safety Statements (S) are listed below:
R35
= causes severe burns
S2
= keep out of the reach of children
S26
= in case of contact with eyes, rinse immediately with plenty of water and seek. medical advice.
S30
= never add water to this product.
S45
= in case of accident or if you feel unwell, seek medical advice immediately.
Symbols according to EC No. 1272/2008 (CLP):
9. General comments
All contents of the BetaPrion® BSE EIA Test Kit are produced under the guidelines of quality
accordingly to the DIN EN ISO 9001:2008 requirements.
11
10.
Instruction for the sample preparation using AJ Roboscreen BSE sample syringe
Introduction
AJ Roboscreen offers a syringe for sampling of 350 ± 50 mg from the Obex region of the brainstem for testing with the
®
BetaPrion BSE EIA test kit.
It is recommended to use this instruction for taking the Obex sample with this syringe. Sampling using the syringe must be
done by professional operators. The competence of the operators must be shown by a monitoring scheme in the lab that
includes a professional training to know the anatomical orientation of the brainstem and the target area.
The syringe is easy to use. The syringe barrel is labelled with 0.1 ml steps (figure 2). Each 0.1 ml represents 100 mg
tissue material. The correct amount of brain tissue to be sampled from the Obex is 350 ± 50 mg. Therefore, the operator
must remove between 0.3 and 0.4 ml of tissue material.
Cuting
wire
Barrel of the syringe
Piston
Figure 2: Scheme of the AJ Roboscreen BSE sample syringe
Histopathological description of the sample region
The preferred sample should be at, or within 1.5 cm anterior to, the Obex (figure 3).
Figure 3:
Brainstem after the removal of the
Cerebellum, from a) dorsal, and b)
lateral aspects.
Recommended levels at which
sections for histopathological
analysis should be taken (OIE
Manual of Diagnostic Tests and
Vaccines for Terrestrial Animals):
A–A = Medulla, at the Obex
B-B = Medulla through caudal
cerebellar peduncles
C-C = midbrain through rostral
colliculi
12
Sampling the rostral Medulla unilaterally for rapid testing must not compromise examination by histological or
immunohistochemical means. The nucleus of the solitary tract (the target area in cattle) is small and lies close to
midline (figure 4). The other early target area , the nucleus of the spinal tract of the trigeminal nerve, lies more
laterally.
Figure 4:
Cross section of the brain-stem at the level of the obex
identifying the key target sites for diagnosis by
histopathology and immunohistochemistry of BSE
(OIE Manual of Diagnostic Tests and Vaccines for
Terrestrial Animals):
-
nucleus of the solitary tract [1]
-
nucleus of the spinal tract of the trigeminal nerve
(V) [2]
-
dorsal nucleus of the vagus [3]
Usage of the syringe
The sampling region must be at or within 1.5 cm anterior to the Obex (figure 5).
Figure 5: Sample area.
sampling sites
Obex Spinal Cord
Before sampling, the length of the brain stem must be determined in order to be sure that the sampling is possible
from the Obex with the syringe. In some brain tissue samples, the Spinal Cord is longer than the syringe. In this case
cut the Spinal Cord with a scalpel, to enable the syringe to reach the sampling area.
Take the syringe with one hand and push the piston to the home position inside the syringe barrel. With the other
hand take the brain stem and fix it on a disposable paper sheet. The caudal end of the brain stem/spinal cord should
be accessible.
Now insert the syringe on the left or right side of the brain stem. One side of the brain stem must remain intact after
sampling. This is reserved for confirmatory testing (figure 6). During insertion of the syringe into one side of the brain
stem take care that it does not cross the midline.
13
Figure 6: The syringe before the insertion into the right side of the brain stem.
Simultaneously, with inserting of the syringe into the tissue withdraw the piston from the syringe in order to generate
a vacuum. This will allow the brain tissue to enter the barrel of the syringe. Press the sampling area with a finger of
the other hand that fixes the brain. Stop the inserting if the end of the sampling area anterior to the Obex is reached
(figure 7).
Figure 7: The syringe has reached the end of the sampling area.
Cut the sample by rotating the barrel two times. Slowly remove the syringe from the brain stem. The tissue sample
should be remained inside the barrel.
Push the piston to remove any possible air gaps from the tip of the syringe. Be sure that all of sample is still inside the
barrel. Push the piston to the next calibration on the syringe.
Take a homogenisation tube and remove the lid. Hold the syringe at the top of the tube.
Carefully push the piston to the middle of the label between 0.3- 0.4 ml and remove about 0.35 ml of tissue material.
Cut the sample by gripping the top of the syringe against the top of the grinding tube.
Precautions
The sample syringes are disposable materials and must be used only once. The used syringe must be
decontaminated and discarded as described below.
Autolysed samples could be taken by the syringe from the region that is identified as the Obex. For this case the
sample should be raised by the syringe up to about 0.35 ml.
Damaged samples should be taken by a scalpel according to the BetaPrion manual.
Health and safety instructions
Wear disposable gloves when handling with reagents and samples from TSE risk materials.
Prions are a unique class of pathogens which exhibits unusual resistance to conventional chemical and physical
decontamination methods. A Biosafety level 3** laboratory is necessary for testing of TSE material.
Use disposible consumable materials whenever possible. For decontamination of TSE positive material autoclaving
at 134°C for 1 hour is recommended.
Surfaces, instruments and liquids that are not autoclaveable must be decontaminated in 2.5% sodium hypochlorite or
1 M NaOH for at least 1 hour.
in 2.5% sodium hypochlorite or 1 M NaOH for at least 1 hour.
Incineration of all decontaminated waste is recommended.
The operator must receive specific training to work with risk material and prions. Decontamination of TSE risk
materials and health and safety instructions must be comply with national legal regulations of the concerned country.
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Contacts
Producer
AJ Roboscreen GmbH
HohmannStraße 7, 04129 Leipzig
E-mail: [email protected]
phone: +49-341-9897340
fax: +49-341-989734199.
Seller
Analytik Jena AG
Konrad-Zuse-Straße 1
07745 Jena
E-mail: [email protected]
phone: +49-3641-77-70
fax: +49-3641-77-92-79
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